ProjectSummary Thelongtermgoalofthisprogramistounderstandhowglycosylationregulatesbiologicalevents, such as cell differentiation and growth control, in development and cancer. We showed previously that MGAT1, the GlcNAc-transferase that initiates complex N-glycan synthesis, is essential for spermatogenesis. Recently, we determined that conditional loss of MGAT1 in spermatogonia leads to premature upregulation of genes normally expressed later in spermatogenesis, and to reduced ERK1/2 signaling that we hypothesize is due, at least in part, tothelossofcomplexN-glycansontheMGAT1targetbasigin.Globaldeletionofbasigingivesa similar block in spermatogenesis, and loss of complex glycans on basigin leads to reduced ERK1/2 signaling. We will test the hypothesis that the N-glycans on basigin promote ERK1/2 signaling and their loss in after MGAT1 removal leads to defective spermatogenesis. We will determine if complex N-glycans mediate Sertoli-spermatid interactions important for spermatogenesis.Ifso,weknowthathybridN-glycansdonotsupportspermatogenesis,andwill determineifbiantennary,complexN-glycanssuffice.SinceMGAT1ispivotaltothegenerationof complex N-glycans, and the absence of N-glycans is desirable in a large variety of different contexts, including to inhibit cancer progression, it has long been desirable to have a small molecule inhibitor of MGAT1. We believe that the time is ripe to succeed in this endeavor. We have shown thatGnT1IP, a physiological inhibitor of MGAT1, forms heteromers with MGAT1 in the Golgi, and identified the C-terminal two amino acids in GnT1IP as separately necessary to inhibit MGAT1. We will determine 3D structures of MGAT1, GNT1IP and MGAT1/GnT1IP heterodimers, with and without these mutations, to determine the mechanism of inhibition. We will use this information to developa TAT-peptide inhibitor that will work in theGolgi. Finally, we will isolate small molecule inhibitor(s) specific for MGAT1 by high throughput screening of chemicallibraries,andbyinsilicodockingof14millioncompoundstoavailablecrystalstructures of MGAT1 versus related glycosyltransferases MGAT2 and POMGNT1. A small molecule inhibitor of MGAT1 will be extremely valuable in many areas of research, for glycosylation engineering of biotherapeutics, as a potential male contraceptive, and for inhibiting cancer progression.